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Ganglioside GD3 induces convergence and synergism of adhesion and hepatocyte growth factor/Met signals in melanomas.

Furukawa K, Kambe M, Miyata M, Ohkawa Y, Tajima O, Furukawa K - Cancer Sci. (2013)

Bottom Line: In this study, we analyzed the effects of GD3 expression on cell signals triggered by hepatocyte growth factor (HGF)/Met interaction and by adhesion to collagen type I (CL-I).When resistance to induced apoptosis by H2O2 was examined, only GD3+ cells treated with both HGF and adhesion to CL-I showed clearly low percentages of dead cells compared with GD3- cells or GD3+ cells treated with either one of the stimulants.Cell growth measured by 5-ethynyl-2' deoxyuridine uptake also showed synergistic effects in GD3+ cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Chubu University College of Life and Health Sciences, Kasugai, Japan; Department of Biochemistry II, Nagoya University Graduate School of Medicine, Nagoya, Japan.

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Increased resistance to apoptosis induced by H2O2 by simultaneous stimulation with hepatocyte growth factor (HGF) and collagen type I (CL-I) in ganglioside GD3+ N1 cells. (A) After incubation for 16 h in serum-free medium, GD3+ cells and GD3− cells were collected. These cells were suspended in FCS-free medium and rotated for 1 h at 37°C. Cell suspensions were plated in CL-I-coated 96-well plates (2.0 × 103 cells/100 μL/well), and added with 10 ng/mL HGF and 5 μM CellEvent Caspase-3/7 Green Detection Reagent. After 30 min incubation at 37°C, the cells were treated with 0 or 1500 μM H2O2 for 4.5 h and apoptotic cells (identified by bright green nuclei caused by activated caspase-3/7) were counted. (B) Cells were harvested and rotated for 1 h at 37°C after serum starvation for 14–16 h. Then cell suspensions were plated in CL-I-coated 96-well plates (2.0–3.0 × 103 cells/100 μL/well) (a), or cultured with HGF (10 ng/mL) in non-coated 1.5 mL tubes (5.0 × 105 cells/1.0 mL) (b), or plated in CL-I-coated 96-well plates (2.0–3.0 × 103 cells/100 μL/well) with simultaneous addition of HGF (10 ng/mL) (c). After 30 min incubation, these cells were treated with different concentrations of H2O2 for 2 or 4.5 h, and apoptotic cells with activated caspase-3/7 showed bright green nuclei. The percentage of apoptotic cells was calculated by the number of cells labeled with bright green in the total number of cells. The percentage of dead cells at the starting point just after seeding cells to plates was subtracted as a baseline from the percentage of apoptotic cells at each time point.
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fig07: Increased resistance to apoptosis induced by H2O2 by simultaneous stimulation with hepatocyte growth factor (HGF) and collagen type I (CL-I) in ganglioside GD3+ N1 cells. (A) After incubation for 16 h in serum-free medium, GD3+ cells and GD3− cells were collected. These cells were suspended in FCS-free medium and rotated for 1 h at 37°C. Cell suspensions were plated in CL-I-coated 96-well plates (2.0 × 103 cells/100 μL/well), and added with 10 ng/mL HGF and 5 μM CellEvent Caspase-3/7 Green Detection Reagent. After 30 min incubation at 37°C, the cells were treated with 0 or 1500 μM H2O2 for 4.5 h and apoptotic cells (identified by bright green nuclei caused by activated caspase-3/7) were counted. (B) Cells were harvested and rotated for 1 h at 37°C after serum starvation for 14–16 h. Then cell suspensions were plated in CL-I-coated 96-well plates (2.0–3.0 × 103 cells/100 μL/well) (a), or cultured with HGF (10 ng/mL) in non-coated 1.5 mL tubes (5.0 × 105 cells/1.0 mL) (b), or plated in CL-I-coated 96-well plates (2.0–3.0 × 103 cells/100 μL/well) with simultaneous addition of HGF (10 ng/mL) (c). After 30 min incubation, these cells were treated with different concentrations of H2O2 for 2 or 4.5 h, and apoptotic cells with activated caspase-3/7 showed bright green nuclei. The percentage of apoptotic cells was calculated by the number of cells labeled with bright green in the total number of cells. The percentage of dead cells at the starting point just after seeding cells to plates was subtracted as a baseline from the percentage of apoptotic cells at each time point.

Mentions: As the phosphorylation of Akt was synergistically enhanced by costimulation with HGF and adhesion to CL-I in GD3+ cells, we examined the role of enhanced Akt activation caused by the combined signals in apoptosis. We treated GD3+ and GD3− cells with H2O2 after stimulation with either adhesion to CL-I or HGF treatment or costimulation with HGF and CL-I at the same time in GD3+ cells and GD3− cells. After adhesion stimulation to CL-I, the percentage of apoptotic cells in total cells was lower in GD3+ cells than in GD3− cells where cells were treated with 1000 or 1500 μM H2O2 for 4.5 h (Fig.7B(a)). When cells were stimulated only by HGF, and subsequently treated with 1000 or 1500 μM H2O2 for 2–4.5 h, the percentage of apoptotic cells in total cells was 40–50% in both GD3+ and GD3− cells (Fig.7B(b)). After costimulation with HGF and CL-I, the percentage of apoptotic cells in total cells was significantly lower in GD3+ cells than in GD3− cells when cells were treated with 1500 μM H2O2 for 2 h, and 1000 and 1500 μM H2O2 for 4.5 h (Fig.7B(c)). Compared with single stimulation using either HGF or adhesion to CL-I, costimulation with both was more effective in GD3+ cells, as the percentage of apoptotic cells in GD3+ cells was less than half that in GD3− cells after treatment with 1500 μM H2O2 for 2 h and 4.5 h (Fig.7B(a,c)). The apoptotic cells (indicated by bright green nuclei) at 4.5 h of H2O2 treatment are shown in Figure7(A). These results suggested that GD3 expression confers resistance to apoptosis with costimulation with HGF and adhesion to CL-I.


Ganglioside GD3 induces convergence and synergism of adhesion and hepatocyte growth factor/Met signals in melanomas.

Furukawa K, Kambe M, Miyata M, Ohkawa Y, Tajima O, Furukawa K - Cancer Sci. (2013)

Increased resistance to apoptosis induced by H2O2 by simultaneous stimulation with hepatocyte growth factor (HGF) and collagen type I (CL-I) in ganglioside GD3+ N1 cells. (A) After incubation for 16 h in serum-free medium, GD3+ cells and GD3− cells were collected. These cells were suspended in FCS-free medium and rotated for 1 h at 37°C. Cell suspensions were plated in CL-I-coated 96-well plates (2.0 × 103 cells/100 μL/well), and added with 10 ng/mL HGF and 5 μM CellEvent Caspase-3/7 Green Detection Reagent. After 30 min incubation at 37°C, the cells were treated with 0 or 1500 μM H2O2 for 4.5 h and apoptotic cells (identified by bright green nuclei caused by activated caspase-3/7) were counted. (B) Cells were harvested and rotated for 1 h at 37°C after serum starvation for 14–16 h. Then cell suspensions were plated in CL-I-coated 96-well plates (2.0–3.0 × 103 cells/100 μL/well) (a), or cultured with HGF (10 ng/mL) in non-coated 1.5 mL tubes (5.0 × 105 cells/1.0 mL) (b), or plated in CL-I-coated 96-well plates (2.0–3.0 × 103 cells/100 μL/well) with simultaneous addition of HGF (10 ng/mL) (c). After 30 min incubation, these cells were treated with different concentrations of H2O2 for 2 or 4.5 h, and apoptotic cells with activated caspase-3/7 showed bright green nuclei. The percentage of apoptotic cells was calculated by the number of cells labeled with bright green in the total number of cells. The percentage of dead cells at the starting point just after seeding cells to plates was subtracted as a baseline from the percentage of apoptotic cells at each time point.
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fig07: Increased resistance to apoptosis induced by H2O2 by simultaneous stimulation with hepatocyte growth factor (HGF) and collagen type I (CL-I) in ganglioside GD3+ N1 cells. (A) After incubation for 16 h in serum-free medium, GD3+ cells and GD3− cells were collected. These cells were suspended in FCS-free medium and rotated for 1 h at 37°C. Cell suspensions were plated in CL-I-coated 96-well plates (2.0 × 103 cells/100 μL/well), and added with 10 ng/mL HGF and 5 μM CellEvent Caspase-3/7 Green Detection Reagent. After 30 min incubation at 37°C, the cells were treated with 0 or 1500 μM H2O2 for 4.5 h and apoptotic cells (identified by bright green nuclei caused by activated caspase-3/7) were counted. (B) Cells were harvested and rotated for 1 h at 37°C after serum starvation for 14–16 h. Then cell suspensions were plated in CL-I-coated 96-well plates (2.0–3.0 × 103 cells/100 μL/well) (a), or cultured with HGF (10 ng/mL) in non-coated 1.5 mL tubes (5.0 × 105 cells/1.0 mL) (b), or plated in CL-I-coated 96-well plates (2.0–3.0 × 103 cells/100 μL/well) with simultaneous addition of HGF (10 ng/mL) (c). After 30 min incubation, these cells were treated with different concentrations of H2O2 for 2 or 4.5 h, and apoptotic cells with activated caspase-3/7 showed bright green nuclei. The percentage of apoptotic cells was calculated by the number of cells labeled with bright green in the total number of cells. The percentage of dead cells at the starting point just after seeding cells to plates was subtracted as a baseline from the percentage of apoptotic cells at each time point.
Mentions: As the phosphorylation of Akt was synergistically enhanced by costimulation with HGF and adhesion to CL-I in GD3+ cells, we examined the role of enhanced Akt activation caused by the combined signals in apoptosis. We treated GD3+ and GD3− cells with H2O2 after stimulation with either adhesion to CL-I or HGF treatment or costimulation with HGF and CL-I at the same time in GD3+ cells and GD3− cells. After adhesion stimulation to CL-I, the percentage of apoptotic cells in total cells was lower in GD3+ cells than in GD3− cells where cells were treated with 1000 or 1500 μM H2O2 for 4.5 h (Fig.7B(a)). When cells were stimulated only by HGF, and subsequently treated with 1000 or 1500 μM H2O2 for 2–4.5 h, the percentage of apoptotic cells in total cells was 40–50% in both GD3+ and GD3− cells (Fig.7B(b)). After costimulation with HGF and CL-I, the percentage of apoptotic cells in total cells was significantly lower in GD3+ cells than in GD3− cells when cells were treated with 1500 μM H2O2 for 2 h, and 1000 and 1500 μM H2O2 for 4.5 h (Fig.7B(c)). Compared with single stimulation using either HGF or adhesion to CL-I, costimulation with both was more effective in GD3+ cells, as the percentage of apoptotic cells in GD3+ cells was less than half that in GD3− cells after treatment with 1500 μM H2O2 for 2 h and 4.5 h (Fig.7B(a,c)). The apoptotic cells (indicated by bright green nuclei) at 4.5 h of H2O2 treatment are shown in Figure7(A). These results suggested that GD3 expression confers resistance to apoptosis with costimulation with HGF and adhesion to CL-I.

Bottom Line: In this study, we analyzed the effects of GD3 expression on cell signals triggered by hepatocyte growth factor (HGF)/Met interaction and by adhesion to collagen type I (CL-I).When resistance to induced apoptosis by H2O2 was examined, only GD3+ cells treated with both HGF and adhesion to CL-I showed clearly low percentages of dead cells compared with GD3- cells or GD3+ cells treated with either one of the stimulants.Cell growth measured by 5-ethynyl-2' deoxyuridine uptake also showed synergistic effects in GD3+ cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Chubu University College of Life and Health Sciences, Kasugai, Japan; Department of Biochemistry II, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Show MeSH
Related in: MedlinePlus